Bacteria and viruses and clinical outcomes of asthma‐bronchiectasis overlap syndrome: A cohort study

Abstract Background Despite the high prevalence of co‐existing bronchiectasis and asthma (asthma‐bronchiectasis overlap syndrome [ABOS]), little is known regarding the dominant pathogens and clinical correlates. Objective To investigate the bacteria and viruses which differentially dominate in ABOS (including its subtypes) compared with bronchiectasis alone, and determine their relevance with bronchiectasis severity and exacerbations. Methods This was a prospective observational cohort study conducted between March 2017 and August 2023. We included 81 patients with ABOS and 107 patients with bronchiectasis alone. At steady‐state baseline, patients underwent comprehensive assessments and sputum collection for bacterial culture and viral detection (quantitative polymerase‐chain‐reaction). Patients were followed‐up to record exacerbation and spirometry. Results Patients with ABOS had significantly higher symptom burden and exacerbation frequency than those with bronchiectasis alone. Despite similar pathogen spectrum, the rate of bacteria–virus co‐detection increased less substantially at acute exacerbations (AE) onset than at steady‐state compared with bronchiectasis alone. Pathogenic bacteria (particularly Pseudomonas aeruginosa) were detected fairly common (exceeding 50%) in ABOS and were associated with greater severity of bronchiectasis when stable and conferred greater exacerbation risks at follow‐up. Viral but not bacterial compositions changed substantially at AE onset compared with clinical stability. Higher blood eosinophil count, moderate‐to‐severe bronchiectasis and non‐atopy were associated with higher odds of bacterial, but not viral, detection (all p < 0.05). Conclusion Detection of bacteria or virus is associated with bronchiectasis severity or clinical outcomes in ABOS. This highlights the importance of integrating sputum microbial assessment for ascertaining the dominant pathophysiology (atopy vs. infection) and longitudinal trajectory prediction in ABOS.


| INTRODUCTION
Both asthma and bronchiectasis are common chronic respiratory diseases (CRDs). 1 For bronchiectasis, the pathological bronchial dilation primarily stems from chronic airway infection and inflammation-the latter has been the shared pathophysiological component of asthma. 2 Depending on the clinical milieu, asthma may develop among some patients with bronchiectasis, while bronchiectasis might develop among some asthmatic patients through the trajectory of progression. 3Bronchiectasis is common among patients with severe asthma.For instance, in a systematic review, the prevalence of asthma in patients with bronchiectasis ranged between 15% and 30.2%.Patients with coexisting bronchiectasis and asthma (asthma-bronchiectasis overlap syndrome [ABOS]) reportedly had a higher exacerbation frequency than those with bronchiectasis alone. 1,4Despite the high prevalence, little is known regarding the pathophysiology of ABOS.Moreover, there is no study that explores the causal relationship between asthma and bronchiectasis.
Chronically inflamed airways have formed a niche for infection with respiratory pathogens.An outgrowth of pathogenic bacteria (e.g.,

Haemophilus influenzae [HI]
) has been implicated in asthma 5 and bronchiectasis 6 and correlates significantly with the disease severity, inflammatory responses and prognosis. 6Apart from bacteria, some viruses have been linked to heightened inflammation and exacerbation of asthma 7,8 and bronchiectasis. 9cumulating evidence has begun to unravel the interactions between bacteria and viruses in CRDs.Rhinovirus detection frequently coincided with bacterial detection, and rhinovirus co-existing with Moraxella catarrhalis or Streptococcus pneumoniae has been associated with greater symptom burden and exacerbation risks in asthmatic children. 10Furthermore, viral detection, isolation of new bacteria, and bacterial isolation plus viral detection have been consistently associated with bronchiectasis exacerbations. 11 hypothesized that some pathogens differentially dominate in ABOS (including its subtypes) compared with bronchiectasis alone.
Here, we elucidated the spectra of bacteria and viruses at clinical stability and exacerbations of ABOS, and their clinical correlates.These may help better appraise the role of bacterial detection and the clinical relevance in ABOS.

| Study participants
We consecutively recruited adults from out-patient clinics between March 2017 and August 2023.Clinically significant bronchiectasis was diagnosed as high-resolution computed tomography (HRCT) manifestations (an inner airway-artery diameter ratio of 1.5 or more, an outer airway-artery diameter ratio of 1.5 or more, a lack of tapering of the airways, and visibility of airways in the periphery) compatible with respiratory symptoms (particularly daily cough, chronic mucopurulent or purulent sputum, a history of exacerbations). 12Eligible patients with ABOS should be in steadystate at baseline -remaining exacerbation-free and had no antibiotics used (except for low-dose macrolides) for >4 weeks.Acute exacerbations (AEs) denoted a significant deterioration of three or more symptoms persisting for at least 48 h that required immediate changes in treatment, according to European Respiratory Society expert consensus. 13Asthma was a physician-diagnosis based on Global Initiative for Asthma guidelines-respiratory symptoms (wheezing, shortness of breath, cough or chest tightness) plus variable expiratory airflow limitation based on the significant bronchodilator response or airway hyperresponsiveness. 1 Severe asthma was defined as asthma requiring level 4/5 treatments to maintain asthma control, or asthma that remained uncontrolled despite the above-mentioned treatments.ABOS was diagnosed among patients with co-existing asthma and bronchiectasis.ABOS

| Study design
This study was divided into two sections.Section 1 was a pro-

| Procedures
At initial visits, we collected clinical information, and performed etiological work-up, spirometry, 15 chest HRCT, 16 fractional exhaled nitric oxide assay (if indicated) and symptom questionnaire which inquired upper and lower airway symptoms (rating the severity with visual analog scale (VAS, range: 0-10]).We rated bronchiectasis severity with Bronchiectasis Severity Index (BSI) 17,18 and E-FACED score. 19We ascertained atopic status according to the total or specific immunoglobulin E titers for allergens (including inhaled allergens, molds, and food proteins) in serum or skin-prick test findings with standard array testing.Blood eosinophil count was stratified into high (>300/μL) or low eosinophil (≤300/μL) subgroups. 1ontaneous sputum collection (aided with chest physiotherapy) was prioritized, or alternatively, induced sputum with 3% saline if sputum yield was insufficient.We performed a quality check and split sputum for differential cell counts, bacterial culture and viral detection with quantitative polymerase-chain-reaction. We defined repeated detection of the same pathogenic bacteria as the isolation of the same bacteria at least twice within a year, at intervals of 3 months or more.
Patients immediately contacted investigators upon significant symptom aggravation.We followed patients via telephone every 2 months and scheduled outpatient clinics every 3-6 months for recording exacerbation history and spirometry.Sputum samples were collected prior to the prescription of antibiotics, and AE samples were collected on the first day of AE.

| Pathogen detection
Laboratory techniques for pathogen detection included bacterial culture and viral detection.We conducted bacterial culture by homogenizing fresh sputum. 20

| Statistical analysis
Sample size was calculated using PASS software version 11.0.7.The proportion of bronchiectasis patients with bacterial isolation and viral detection was 59.8% and 11.4% at stable visits, and 61.9% and 29.0% at AEs, respectively. 11Therefore, we estimated to recruit 102 ABOS patients with two-sided significance of 0.05 and power of 80%, assuming 20% dropout at follow-up.
Data were presented as mean � standard deviation (SD), median (interquartile range [IQR]) for continuous variables, and counts (proportion) for categorical variables.We explored the association between pathogen detection and the odds of AEs compared with stable visits by using generalized estimating equations, with a logit link and repeated observations among study participants.We analyzed continuous variables with t-test, analysis-of-variance, Mann-Whitney, or Kruskal-Wallis test, depending on the variable distribution, and compared categorical variables with Chi-square or Fisher exact test.Univariate Logistic analysis was conducted to explore the factors of positive sputum culture or repeated detection of the same pathogenic bacteria, and detection or repeated detection of PA, with results being reported as odds ratio (OR) and 95% confidence interval (95% CI).We employed multiple Logistic regression models to assess confounders.Data with p ≤ 0.10 were entered into multivariate analysis as covariates using the backward selection technique.The variables adjusted consisted of the age, 24-h sputum, allergic rhinitis, Reiff score, FEV 1 pred%, FEV 1 /FVC, the number of bronchiectatic lobes, blood neutrophil count, blood eosinophil count, and sputum neutrophil count (percentage).Missing data were not imputed.The future risk of AE was analyzed with Kaplan-Meier model and compared with the log-rank test.SPSS (version 23.0) and Graphpad Prism (version 5.0) were used for statistical analyses.p < 0.05 was considered statistically significant.

| Baseline characteristics associated with pathogen detection in ABOS
Of 114 patients with ABOS screened, 81 were enrolled.The reasons of exclusion are demonstrated in Figure 1.Eighty one Patients with ABOS were divided into bacterial culture positive and negative groups, or PA group and non-PA group.After excluding 10 patients with missing viral detection status at baseline, 75 patients with ABOS were stratified into viral positive and negative groups.Bacterial detection was associated with a longer disease duration, higher daily sputum yield, higher BSI and the modified Reiff scores, more severe airflow limitation, and a more frequent use of muscarinic antagonists.
Sputum culture negative was more likely to be associated with previous wheezing episodes, allergic rhinitis, and blood eosinophilia (Table 1).The proportion of patients with allergy history (e.g.allergic rhinitis) was significantly higher in the PA-negative group than in the PA-positive group.At baseline, ABOS patients with PA or any bacteria detected had more severe bronchiectasis.However, the clinical characteristics did not differ significantly between the viral positive and negative groups at baseline (Table S1).In light of the differential bacterial spectrum between asthma and bronchiectasis, we interrogated the factors predicting the detection of any pathogenic bacteria at baseline when clinically stable among patients with ABOS.To this end, we probed the association between different clinical variables and the detection of pathogenic bacteria using a multivariable logistic regression model.Because the BSI and E-FACED scores included repeated detection of any pathogenic bacteria or PA, we used the modified Reiff score and FEV 1 pred% to analyze the risk factors for bacterial detection at baseline.In the multivariable logistic regression model, higher FEV 1 pred% (OR: 0.95, 95% CI: 0.91-0.99,p = 0.010) was associated with no bacterial detection.A longer duration of symptom onset (OR: 1.06, 95% CI: 1.02-1.11,p = 0.006) was significantly associated with PA detection in ABOS.A longer duration of symptom onset (OR: 1.06, 95% CI: 1.01-1.11,p = 0.009) and sputum neutrophilia (OR: 1.14, 95% CI: 1.01-1.29,p = 0.031) were significantly associated with the repeated PA detection when clinically stable (Table 2).   of 11 0.86-3.82).However, no significant difference in this metric was identified between the PA and non-PA groups (Figure 2).Note: Also adjusted: the duration of symptom onset a,b,c , wheezing a , atopy a , ex-smoker a , sputum eosinophils(%) a , C-reactive protein b , No. Of patients hospitalized in the previous year b .

| Patient recruitment
Abbreviation: FEV1 pred%, the forced expiratory volume in one second percentage predicted.

of 11
- steady-state (both p > 0.05) (Figure 3). in patients with bronchiectasis alone were herpes simplex virus, coronavirus and rhinovirus.In the univariate logistic regression model, pathogen detection did not differ significantly between patients with ABOS and bronchiectasis alone (Table 3).

| Clinical characteristics differentiating AEs with different pathogens
Having demonstrated the symptoms and laboratory test findings that differentiated bacterial from viral detection in our previous study, we then interrogated whether similar conclusions would apply to ABOS.
According to the virus detection status at AEs (Figure 3 AEs, neither upper nor lower airway symptoms differed when comparing these two strata (Table S5).Symptom questionnaires were obtained from 38 (71.7%)AEs, blood routine test from 27 (50.9%) AEs, and C-reactive protein levels from 26 (49.1%)AEs.Again, none of these metrics differed significantly between these two strata (Figure S2 and S3).However, compared with other pathogen strata, patients in viral detection without bacterial detection strata more frequently reported cough, breathlessness and wheeze at AE onset.

| Subgroup analysis of pathogen detection at steady-state
Finally, we compared the pathogen detection rate across different strata, particularly the blood eosinophil count and bronchiectasis severity-the core metrics determining the heterogeneity of ABOS.
Twenty four samples corresponded to the low while 178 samples to high blood eosinophil count strata, the latter of which yielded a lower detection rate of bacteria but not viruses.Sixty seven samples were collected from patients with mild bronchiectasis and 135 from patients with moderate-severe bronchiectasis, the latter of which was associated with a higher detection rate of bacteria, but not viruses.One hundred and eighty one.samples were derived from NSA-ABOS and 21 from SA-ABOS.No significant differences in the detection rate of bacteria or viruses were identified.Finally, 57 samples were derived from atopic patients and 120 from non-atopic patients.Patients in the non-atopic stratum yielded a higher detection rate of bacteria with or without viruses (p = 0.010), but not viruses alone (Figure 3).

| DISCUSSION
Our study has evaluated for the first time, among patients with ABOS, the association between the spectrum of bacteria and/or viruses at steady-state and AE onset and the clinical characteristics.

F I G U R E 2
The risk of bronchiectasis exacerbations between ABOS patients with or without pathogen detection during the 2year longitudinal follow-up.Annual bronchiectasis exacerbation frequency (a,c,e).Proportion of patients free from bronchiectasis exacerbation (b,d,f).PA, Pseudomonas aeruginosa.
Bacteria (including PA) were detected fairly common (exceeding 50%), which was associated with greater severity of bronchiectasis.
Apart from the variable lung function dynamics, baseline bacterial detection conferred greater exacerbation risks at follow-up.We also found notable changes in viral but not bacterial detection rate at AE onset compared with steady-state.Higher blood eosinophil count, moderate-to-severe bronchiectasis and non-atopy were associated with bacterial but not viral detection.
Bacterial infection has been implicated in various CRDs.Both HI and PA are dominant species in bronchiectasis. 21Despite the low detection rate, 22 the outgrowth of Proteobacteria (e.g.Haemophilus and Pseudomonas spp), as detected with 16srRNA sequencing, was more frequently detected in asthmatic patients compared with healthy controls. 15Our study has shown a high bacterial detection rate in ABOS, which correlated with bronchiectasis severity.Our findings were consistent with previous The detection rate and composition of bacteria and viruses at steady-state and exacerbation onset of ABOS.ABOS, asthmabronchiectasis overlap syndrome; AE, acute exacerbations of bronchiectasis; BþVþ, both bacteria and viruses detected; BþV−, any pathogenic bacteria detected but no viruses detected; B−Vþ, viruses detected but no pathogenic bacteria detected; B−V−, no bacteria and viruses detected; Bx, bronchiectasis; NSA-ABOS, ABOS with non-severe asthma; SA-ABOS, ABOS with severe asthma.New-occurrencebacteria denotes sputum culture switching from negative to positive, or from one pathogenic bacterium to other pathogenic bacterium.
T A B L E 3 Risk factors for pathogen detection at baseline visit and acute exacerbations of bronchiectasis visits among patients with ABOS.

Patients with ABOS Factors Univariate analysis OR (95% CI) p
Baseline reports, which documented that bacterial (esp.4][25][26] Furthermore, a longer duration of symptom onset and higher BSI were associated with bacterial (esp. PA) detection in ABOS, which partly mirrored those of published studies among patients who had predominantly bronchiectasis alone. 26,27This implied a notable similarity of bacterial detection between ABOS and bronchiectasis alone.
Among patients with ABOS, bacterial detection was associated with less prominent asthma-related characteristics, including a lower likelihood of allergic disease family history and current allergic rhinitis, lower blood eosinophil counts, and requiring less corticosteroid treatment.This suggested a counterbalance between atopy and airway infection in bronchiectasis.We have demonstrated a more prominent airflow limitation and a more frequent and accelerated course of AE in patients with bacterial detection.At baseline, patients with ABOS had more frequently received inhaled medications, higher prior exacerbation frequency and more severe airflow limitation.Interestingly, patients with ABOS demonstrated some improvement in airflow limitation if adequately treated during follow-up.This differed from findings among patients with bronchiectasis alone, who had progressive lung function decline. 28,29Therefore, co-existing asthma and the targeted treatment might be important modifiers of lung function trajectory of ABOS.
Accumulating evidence has unveiled the association between bacteria and/or viruses and AEs, the events with major clinical implications , . 14,30Previous studies have identified a significant association between viral, but not bacterial, detection and AE onset in patients with bronchiectasis alone. 14,20,31,32In ABOS, although both PA and HI were common, they were not directly associated with AE onset.Unlike findings in bronchiectasis alone, we showed no association between the detection of new bacteria and AE onset in ABOS.
Similar to bronchiectasis alone, both bacteria (particularly PA) and viruses were significantly associated with ABOS at AE onset.
Although caution for data interpretation should be exercised given the small sample sizes, the roles of bacteria cannot be readily extrapolated from bronchiectasis alone.
Despite the minor roles of bacteria at AE onset, viruses played crucial roles at AE onset in ABOS.Our findings of the detection rate and spectrum of viruses mirrored those in bronchiectasis alone. 14,20,32A concerning issue is the interaction between bacterial and viral detection.[35] However, the association between co-detection of bacteria and viruses was further stratified by the presence (severe asthma-ABOS [SA-ABOS]) or absence (non-severe asthma [(NSA-ABOS]) of severe asthma.The key exclusion criteria were active tuberculosis, malignancy, eosinophilic granulomatosis with polyangiitis, allergic bronchopulmonary aspergillosis, traction bronchiectasis, insufficient sputum yield, and pregnancy or lactation.Ethics approval was obtained from the Ethics Committee of The First Affiliated Hospital of Guangzhou Medical University (Medical Ethics [2012] the 33 th ; Medical Ethics [2020] the 156 th ).All patients signed written informed consent.
spective observational cohort study (May 2018 to August 2023) investigating bacterial and viral detection in steady-state ABOS and the clinical correlates.Section 2 was an extended study which added historical sputum samples collected since March 2017 (with paired bacterial and viral detection data), 14 which analyzed bacterial and viral detection at AE onset of ABOS.Records of AE onset were incomplete between March 2017 and April 2018, because some patients were lost to follow-up.Therefore, the association between bacterial detection and future risk of AE was analyzed in Section 1 only.
ZHANG ET AL.
3.1.3| Baseline pathogen detection and longitudinal clinical outcomesWe next explored whether pathogen detection at baseline could predict the clinical outcomes during longitudinal follow-up.During the median follow-up of 25.3 (IQR:14.2) months, 81 patients with ABOS reported 137 AE episodes (17 requiring hospitalization).53 patients had paired spirometric reassessment.The forced expiratory volume in one second percentage predicted (FEV 1 pred%) improved considerably from baseline to follow-up (mean: 56.4% vs. 62.0%, p < 0.001), which did not differ significantly when stratified by bacterial (including PA and virus) detection status (p > 0.05) (FigureS1).At baseline, the annualized frequency of AE did not differ significantly when stratified by bacterial (including PA and virus) detection status (p > 0.05).Patients with bacterial detection at baseline had a significantly shorter time to the first AE than those without (median: 7 vs. 13 months, HR: 1.73, 95% CI: 1.05-2.86).Patients with viral detection at baseline also had a significantly shorter time to the first AE than those without (median: 4 vs. 9 months, HR: 1.81, 95% CI: F I G U R E 1 Flow chart of patient recruitment.ABOS, asthma-bronchiectasis overlap syndrome; AE, acute exacerbations of bronchiectasis; Bx, bronchiectasis alone.T A B L E 1 Baseline clinical characteristics in ABOS patients with or without bacterial detection when clinically stable. ), we classified patients with ABOS into virus-positive (n = 11) versus virusnegative (n = 27) strata, or into viral detection without bacterial detection (VþB−, n = 6) versus other pathogen strata (n = 32).At and AE onset has missed the statistical significance, again possibly because of the limited sample sizes.Some limitations should be considered.The small sample size was a clear limitation and the analysis performed was exploratory.Some patients dropped out because of the long distance from residential places to the study site and the COVID-19 outbreak.Replication of these correlation analyses and subsequent findings in a larger sample is required with subgroup study analysis considering the dominant taxa of the airway microbiome.We have recruited patients from outpatient clinics, decreasing the generalizability of our findings to other hospitalized patients.Because of the sufficient sputum yield for routine bacterial culture, we did not enroll patients with asthma alone as a control group herein.We have only included patients with productive cough at baseline, and repeated pathogen detection cannot be performed among all patients.Furthermore, eosinophils were not measured in patients receiving oral corticosteroids and nontuberculous mycobacteria culture was not performed.The microbial compositions cannot be addressed by sputum culture alone, and sequencing findings will be reported separately.Because of the lack of existing definitions, AE of ABOS was mainly defined based on the criteria for that of bronchiectasis instead of asthma.However, discriminating the attributable causes of the aggravated symptoms is impractical given the overlapping clinical manifestations of asthma and bronchiectasis.In summary, bacterial detection at steady-state correlates with disease severity and future risks of progression in ABOS.Greater attention should be paid to viral detection at AE onset.Our study has highlighted the importance of integrating sputum microbial assessment into clinical practice for ascertaining the dominant pathophysiology (atopy vs. infection) and longitudinal trajectory prediction in ABOS.

Positive sputum culture (n = 45) Negative sputum culture (n = 36) p Value PA positive (n = 31) PA negative (n = 50) p Value
Seventy eight patients had undergone sputum cytology examination, 74 patients had undergone total immunoglobulin E testing, 74 patients had undergone fractional exhaled nitric oxide testing, 77 patients had undergone C-reactive protein testing.No. of bronchiectatic lobes: a metric reflecting the radiological extension of bronchiectasis.Risk factors for bacterial detection or repeated detection in ABOS at baseline.
patients with ABOS provided 202 sputum samples from steady-state visits and 53 from AE visits (83 historical samples from steady-state visits and 28 from AE visits).Each patient provided a median of 3.4 samples.Among patients who provided multiple samples, the median interval from the earliest to the latest sampling was 29.2 (IQR: 12.8) months.The 75 patients reported 136 AEs during this time interval (53 AEs with hospital visits).The 107 patients with bronchiectasis alone provided 226 sputum samples from steady-state visits and 69 from AE visits (median: two samples per patient).Patient recruitment is shown in Figure1.We next analyzed the pathogen spectrum of ABOS by comparing it with bronchiectasis alone.First, we focused on the bacterial detection rate and the bacterial spectrum.The detection rate of bacteria alone (45.3% vs. 52.5%)or in combination with virus (18.9% vs. 11.9%)didnot increase significantly at AE onset compared with T A B L E 1 (Continued) Abbreviations: FEV 1 , forced expiratory volume in one second; FVC, forced vital capacity. a b E-FACED score: an integrated clinical severity metric.cHRCT Reiff score: a metric reflecting the radiological severity of bronchiectasis.d